Biochemistry, 4th Edition P48 pptx

Only about half of the common amino acid residues that is, His, Cys, Asp, Glu, Arg, Lys, Tyr, Ser, Thr, Asn, and Gln engage directly in catalytic effects in enzyme active sites.. In this

Initial step of thermolysin reaction

Glu

N H C

O

N H C

O–

H O H O–

C

HO

O

Glu

OH C O

FIGURE 14.14 Thermolysin is an endoprotease (that is,

it cleaves polypeptides in the middle of the chain) with

a catalytic Zn2ion in the active site The Zn2ion stabi-lizes the buildup of negative charge on the peptide car-bonyl oxygen, as a glutamate residue deprotonates wa-ter, promoting hydroxide attack on the carbonyl carbon Thermolysin is found in certain laundry detergents, where it is used to remove protein stains from fabrics.

peptidase (see Chapter 5) contains an active site Zn2, which facilitates

deprotona-tion of a water molecule in this manner

Mechanisms?

The balance of this chapter will be devoted to several classic and representative

en-zyme mechanisms, including the serine proteases, the aspartic proteases, and

cho-rismate mutase Both the serine proteases and the aspartic proteases use general

A DEEPER LOOK

How Do Active-Site Residues Interact to Support Catalysis?

Only about half of the common amino acid residues (that is, His,

Cys, Asp, Glu, Arg, Lys, Tyr, Ser, Thr, Asn, and Gln) engage directly

in catalytic effects in enzyme active sites These polar and charged

residues provide a relatively limited range of catalytic capabilities

They can act as nucleophiles, facilitate substrate binding, and

sta-bilize transition states It has been estimated that up to 75% of the

steps in enzyme mechanisms involve a simple proton transfer Is

this enough to explain the dramatic catalytic power of enzymes?

Or might there be other phenomena at work?

Janet Thornton and Alex Gutteridge have analyzed residue

in-teractions at the active sites of 191 different enzymes In this group

of enzymes, each polar catalytic residue interacts with (on

aver-age) 2.3 other polar residues in the active site, whereas

noncat-alytic, buried polar residues have, on average, interactions with

only 1.2 other polar residues This suggests that some of the

inter-actions between catalytic and noncatalytic residues are functional

in some way At the same time, in only 88 of the enzymes does the

key catalytic residue have a direct interaction with a second

cat-alytic residue, indicating that most catcat-alytic residues do not

re-quire direct interactions with other catalytic residues to be active

The catalytic capacities of polar and charged residues can be

in-fluenced by other polar and charged residues at the active site and

even by hydrophobic residues The so-called secondary, or

non-catalytic, residues at the active site play interesting roles:

• Raising or lowering catalytic residue pK a values through

electrosta-tic or hydrophobic interactions In aldoketoreductase, an

Asp–Lys pair facilitates general acid–base catalysis, with Lys84

lowering the pKaof Tyr58so that it can donate a proton to the

substrate On the other hand, nearby hydrophobic residues

can provide a nonpolar environment that tends to raise the

pKavalues of acidic residues (such as Asp or Glu) and to lower

the pKavalues of basic residues (such as lysine and arginine)

Hydrophobic environments can change pKavalues by as much

as 5 or 6 pH units

• Orientation of catalytic residues, as will be seen in the serine

proteases, where Asp102orients His57(see Figure 14.21)

• Charge stabilization, as will be seen in chorismate mutase, where

active-site arginines stabilize negatively charged carboxyl groups on the substrate (see Figures 14.31 and 14.33)

• Proton transfers via hydrogen tunneling In such quantum

me-chanical tunneling, the proton transfer is accomplished by molecular motions that lead to degeneracy of a pair of local-ized proton vibrational states (Figure 14.13) Proton tunneling can be facilitated by nearby molecular motions of secondary residues coupled to the motion and vibration of the bonds in question David Leys has shown that aromatic amine dehydro-genase probably accomplishes catalysis by coupling local mo-tions (of two secondary residues, C171and T172) to the vibra-tional states involved in a proton transfer reaction with D128, as shown here

Asp 128

Cys171

Thr 172

Oxidized Trp 109

(cofactor)

䊱 Closeup of the crystal structure of aromatic amine dehydrogenase,

showing the relationship of Asp 128 , Thr 172 , and Cys 171 N atoms are blue;

O atoms are red; C atoms are green; S atom is gold (pdb id  2AH1).

acid–base catalysis chemistry; the serine proteases also employ a covalent catalysis strategy Chorismate mutase, on the other hand, uses neither of these and depends instead on the formation of a NAC to carry out its reaction These particular cases are well understood, because the three-dimensional structures of the enzymes and the bound substrates are known at atomic resolution and because great efforts have been devoted to kinetic and mechanistic studies They are important because they represent reaction types that appear again and again in living systems and because they demonstrate many of the catalytic principles cited previously Enzymes are the catalytic machines that sustain life, and what follows is an intimate look at the inner workings of the machinery

Serine Proteases Serine proteases are a class of proteolytic enzymes whose catalytic mechanism

is based on an active-site serine residue Serine proteases are one of the

best-characterized families of enzymes This family includes trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, tissue plasminogen activator, and other related enzymes.

The first three of these are digestive enzymes and are synthesized in the pancreas

and secreted into the digestive tract as inactive proenzymes, or zymogens Within the

digestive tract, the zymogen is converted into the active enzyme form by cleaving off

a portion of the peptide chain Thrombin is a crucial enzyme in the blood-clotting cascade, subtilisin is a bacterial protease, and plasmin breaks down the fibrin poly-mers of blood clots Tissue plasminogen activator (TPA) specifically cleaves the

proenzyme plasminogen, yielding plasmin Owing to its ability to stimulate breakdown

of blood clots, TPA can minimize the harmful consequences of a heart attack, if ad-ministered to a patient within 30 minutes of onset Finally, although not itself a

pro-tease, acetylcholinesterase is a serine esterase and is related mechanistically to the serine

proteases It degrades the neurotransmitter acetylcholine in the synaptic cleft be-tween neurons

The Digestive Serine Proteases

Trypsin, chymotrypsin, and elastase all carry out the same reaction—the cleavage of a peptide chain—and although their structures and mechanisms are quite similar, they display very different specificities Trypsin cleaves peptides on the carbonyl side of the basic amino acids, arginine or lysine (see Table 5.2) Chymotrypsin prefers to cleave

on the carbonyl side of aromatic residues, such as phenylalanine and tyrosine Elastase

is not as specific as the other two; it mainly cleaves peptides on the carbonyl side of small, neutral residues These three enzymes all possess molecular weights in the range of 25,000, and all have similar sequences (Figure 14.15) and three-dimensional structures The structure of chymotrypsin is typical (Figure 14.16) The molecule is el-lipsoidal in shape and contains an -helix at the C-terminal end (residues 230 to 245) and several -sheet domains Most of the aromatic and hydrophobic residues are buried in the interior of the protein, and most of the charged or hydrophilic residues are on the surface Three polar residues—His57, Asp102, and Ser195—form what is

known as a catalytic triad at the active site (Figure 14.17) These three residues are

conserved in trypsin and elastase as well The active site is actually a depression on the surface of the enzyme, with a pocket that the enzyme uses to identify the residue for which it is specific (Figure 14.18) Chymotrypsin, for example, has a pocket sur-rounded by hydrophobic residues and large enough to accommodate an aromatic side chain The pocket in trypsin has a negative charge (Asp189) at its bottom, facili-tating the binding of positively charged arginine and lysine residues Elastase, on the other hand, has a shallow pocket with bulky threonine and valine residues at the opening Only small, nonbulky residues can be accommodated in its pocket The backbone of the peptide substrate is hydrogen bonded in antiparallel fashion to residues 215 to 219 and bent so that the peptide bond to be cleaved is bound close to His57and Ser195

C

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160 170 180 190 200 210 220 230

240 245

His Asp

Ser

N

C

10 20 30 40 50 60 70 80

90

100

110

120

130

140

150

160 170 180 190 200 210 220 230

240 245

Ser

N

C

20 30 40 50 60 70 80

90

100

110

120

130

140

150

160 170 180 190 200 210 220 230

240 245

Ser

S

S

S

S

S S

S

S S

S

S

S S

S

S

S

S

S

S

S

S S S

S

S

S

FIGURE 14.15 Comparison of the amino acid sequences

of chymotrypsinogen, trypsinogen, and elastase Each circle represents one amino acid Numbering is based

on the sequence of chymotrypsinogen Filled circles in-dicate residues that are identical in all three proteins Disulfide bonds are indicated in orange The positions of the three catalytically important active-site residues (His 57 , Asp 102 , and Ser 195 ) are indicated.

FIGURE 14.16 Structure of chymotrypsin (white) in a complex with eglin C (blue ribbon structure), a

target protein The residues of the catalytic triad (His 57 , Asp 102 , and Ser 195 ) are highlighted His 57 (red)

is flanked by Asp 102 (gold) and by Ser 195 (green) The catalytic site is filled by a peptide segment of

eglin Note how close Ser 195 is to the peptide that would be cleaved in the chymotrypsin reaction (pdb

id  1ACB).

Ser 195

N HN

H

C C C

C

C C

O

O

O

N N

H

HO His 57

C

Asp102

FIGURE 14.17 The catalytic triad of chymotrypsin.

The Chymotrypsin Mechanism in Detail: Kinetics

Much of what is known about the chymotrypsin mechanism is based on studies of the

hydrolysis of artificial substrates—simple organic esters, such as p-nitrophenylacetate (Figure 14.19) p-Nitrophenylacetate is an especially useful model substrate, because

the nitrophenolate product is easily observed, owing to its strong absorbance at 400

nm When large amounts of chymotrypsin are used in kinetic studies with this

sub-strate, a rapid initial burst of p-nitrophenolate is observed (in an amount

approximately equal to the enzyme concentration), followed by a much slower, linear rate of nitrophenolate release (Figure 14.20) Observation of a burst, followed by slower, steady-state product release, is strong evidence for a multistep mechanism, with

a fast first step and a slower second step

In the chymotrypsin mechanism, the nitrophenylacetate combines with the

en-zyme to form an ES complex This is followed by a rapid step in which an acyl-enen-zyme intermediateis formed, with the acetyl group covalently bound to the very reactive Ser195 The nitrophenyl moiety is released as nitrophenolate (Figure 14.20), account-ing for the burst of nitrophenolate product Attack of a water molecule on the acyl-enzyme intermediate yields acetate as the second product in a subsequent, slower

step The enzyme is now free to bind another molecule of p-nitrophenylacetate, and

Elastase Chymotrypsin

Trypsin

FIGURE 14.18 The substrate-binding pockets of trypsin

(pdb id  2CMY), chymotrypsin (pdb id  1ACB), and

elastase (pdb id  3EST) Asp 189 (aqua) coordinates Arg

and Lys residues of peptide substrates in the trypsin

binding pocket Val 216 (purple) and Thr 226 (green) make

the elastase binding pocket shallow and able to

accom-modate only small, nonbulky residues.

C

O

NO2 O

H3C

p-Nitrophenylacetate

FIGURE 14.19 Chymotrypsin cleaves simple esters, in

addition to peptide bonds p-Nitrophenylacetate has

been used in studies of the chymotrypsin mechanism.

E

C O

NO2

NO2

O–

O

H2O

O

–O

CH3

+

H+

H+

Time Lag

Burst

Acetate

Steady-state release

p-Nitrophenolate

(b) (a)

FIGURE 14.20 Burst kinetics observed in the

chy-motrypsin reaction (a) A burst of nitrophenolate

(b, first step) is followed by a slower, steady-state

release After an initial lag period, acetate release

(b, second step) is observed This kinetic pattern

is consistent with rapid formation of an

acyl-en-zyme intermediate (and the burst of

nitropheno-late) The slower, steady-state release of products

corresponds to rate-limiting breakdown of the

acyl-enzyme intermediate.

the p-nitrophenolate product produced at this point corresponds to the slower,

steady-state formation of product in the upper right portion of Figure 14.20 In this

mechanism, the release of acetate is the rate-limiting step and accounts for the

ob-servation of burst kinetics—the pattern shown in Figure 14.20.

The Serine Protease Mechanism in Detail: Events at the Active Site

A likely mechanism for peptide hydrolysis is shown in Figure 14.21 As the backbone

of the substrate peptide binds adjacent to the catalytic triad, the specific side chain

fits into its pocket Asp102 of the catalytic triad positions His57 and immobilizes it

through a hydrogen bond as shown In the first step of the reaction, His57acts as a

general base to withdraw a proton from Ser195, facilitating nucleophilic attack by

Ser195on the carbonyl carbon of the peptide bond to be cleaved This is probably a

concerted step, because proton transfer prior to Ser195attack on the acyl carbon would

leave a relatively unstable negative charge on the serine oxygen In the next step,

donation of a proton from His57to the peptide’s amide nitrogen creates a

proto-nated amine on the covalent, tetrahedral intermediate, facilitating the subsequent

bond breaking and dissociation of the amine product The negative charge on the

peptide oxygen is unstable; the tetrahedral intermediate is short lived and rapidly

breaks down to expel the amine product The acyl-enzyme intermediate that results

is reasonably stable; it can even be isolated using substrate analogs for which further

reaction cannot occur With normal peptide substrates, however, subsequent

nucleophilic attack at the carbonyl carbon by water generates another transient

tetrahedral intermediate (Figure 14.21g) His57acts as a general base in this step,

accepting a proton from the attacking water molecule The subsequent collapse of

the tetrahedral intermediate is assisted by proton donation from His57to the serine

oxygen in a concerted manner Deprotonation of the carboxyl group and its

de-parture from the active site complete the reaction as shown

Until recently, the catalytic role of Asp102 in trypsin and the other serine

pro-teases had been surmised on the basis of its proximity to His57in structures obtained

from X-ray diffraction studies, but it had never been demonstrated with certainty in

physical or chemical studies As can be seen in Figure 14.16, Asp102is buried at the

active site; it is normally inaccessible to chemical modifying reagents In 1987,

Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis

(see Chapter 12) to prepare a mutant trypsin with an asparagine in place of Asp102

This mutant trypsin possessed a hydrolytic activity with ester substrates only

1/10,000 that of native trypsin, demonstrating that Asp102is indeed essential for

catalysis and that its ability to immobilize and orient His57by formation of a

hydro-gen bond is crucial to the function of the catalytic triad

The serine protease mechanism relies in part on a low-barrier hydrogen bond

In the free enzyme, the pKavalues of Asp102and His57are very different, and the

H bond between them is a weak one However, donation of the proton of Ser195to

His57lowers the pKaof the protonated imidazole ring so it becomes a close match

to that of Asp102, and the H bond between them becomes an LBHB The energy

re-leased in the formation of this LBHB is used to facilitate the formation of the

sub-sequent tetrahedral intermediate (Figure 14.21c, g)

The Aspartic Proteases

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known

as aspartic proteases These enzymes are active at acidic (or sometimes neutral) pH,

and each possesses two aspartic acid residues at the active site Aspartic proteases

carry out a variety of functions (Table 14.3), including digestion (pepsin and

chy-mosin), lysosomal protein degradation (cathepsin D and E), and regulation of blood

pressure (renin is an aspartic protease involved in the production of angiotensin, a

hor-mone that stimulates smooth muscle contraction and reduces excretion of salts and

fluid) The aspartic proteases display a variety of substrate specificities, but normally

they are most active in the cleavage of peptide bonds between two hydrophobic

(a)

Asp 102

His 57

O

Ser 195

H

NH R'

C O HN R

C

Asp102

His57

O

Ser195

H

R'

C O R

Binding of substrate

Formation of covalent ES complex

C

Asp102

N

LBHB

His 57

O

Ser 195

H

R'

C O–

R

+

Proton donation

by His 57

C

Asp102

His 57

O

Ser 195

R'

C O–

R +

C—N bond cleavage

NH

NH2

NH NH NH

O

Ser 195 R'

C O

NH

R

NH 2

Release of amino product

C

Asp102

His 57

O

Ser195

C

NH O

Nucleophilic attack by water

Ser 195

H

R'

C O–

+

NH

R'

O H

Collapse of tetrahedral intermediate

O H

H

C

Asp102

His 57

O

Ser 195

R'

C O

NH

Carboxyl product release

O H

Ser195

R'

NH –O

Substrate

NH

C

Asp 102

His57

C

Asp 102

His 57

O

(b)

(i)

C

Asp 102

N

LBHB

H

His57

FIGURE 14.21 A detailed mechanism for the chymotrypsin reaction Note the low-barrier hydrogen bond (LBHB) in

(c) and (g).

A DEEPER LOOK

Transition-State Stabilization in the Serine Proteases

X-ray crystallographic studies of serine protease complexes with

transition-state analogs have shown how chymotrypsin stabilizes

the tetrahedral oxyanion transition states [structures (c) and (g)

in Figure 14.21] of the protease reaction The amide nitrogens

of Ser195and Gly193form an “oxyanion hole” in which the

sub-strate carbonyl oxygen is hydrogen bonded to the amide NOH

groups

Formation of the tetrahedral transition state increases the

in-teraction of the carbonyl oxygen with the amide NOH groups in

two ways Conversion of the carbonyl double bond to the longer

tetrahedral single bond brings the oxygen atom closer to the

amide hydrogens Also, the hydrogen bonds between the charged

oxygen and the amide hydrogens are significantly stronger than

the hydrogen bonds with the uncharged carbonyl oxygen

Transition-state stabilization in chymotrypsin also involves the

side chains of the substrate The side chain of the departing amine

product forms stronger interactions with the enzyme upon

forma-tion of the tetrahedral intermediate When the tetrahedral

inter-mediate breaks down (Figure 14.21d and h), steric repulsion

be-tween the product amine group and the carbonyl group of the

acyl-enzyme intermediate leads to departure of the amine product

–

The oxyanion hole

The oxyanion hole

Gly 193

Ser 195

Gly 193

Ser 195

䊳 The “oxyanion hole” of chymotrypsin stabilizes the tetrahedral

oxy-anion intermediate of the mechanism in Figure 14.21.

Pepsin* Stomach Digestion of dietary protein

Chymosin† Stomach Digestion of dietary protein

Cathepsin D Spleen, liver, and many Lysosomal digestion of proteins

other animal tissues Renin‡ Kidney Conversion of angiotensinogen

to angiotensin I; regulation of blood pressure

HIV-protease§ AIDS virus Processing of AIDS virus proteins

Northrop established that enzyme activity comes from proteins.

for thousands of years, in a gastric extract called rennet, in the making of cheese.

TABLE 14.3 Some Representative Aspartic Proteases

amino acid residues The preferred substrates of pepsin, for example, contain aro-matic residues on both sides of the peptide bond to be cleaved

Most aspartic proteases are composed of 323 to 340 amino acid residues, with molecular weights near 35,000 Aspartic protease polypeptides consist of two ho-mologous domains that fold to produce a tertiary structure composed of two simi-lar lobes, with approximate twofold symmetry (Figure 14.22) Each of these lobes or domains consists of two -sheets and two short -helices The two domains are bridged and connected by a six-stranded, antiparallel -sheet The active site is a deep and extended cleft, formed by the two juxtaposed domains and large enough

to accommodate about seven amino acid residues The two catalytic aspartate residues, residues 32 and 215 in porcine pepsin, for example, are located deep in the center of the active site cleft The N-terminal domain forms a “flap” that extends over the active site, which may help to immobilize the substrate in the active site

On the basis, in part, of comparisons with chymotrypsin, trypsin, and the other ser-ine proteases, it was at first hypothesized that aspartic proteases might function by for-mation of covalent enzyme–substrate intermediates involving the active-site aspartate residues However, all attempts to trap or isolate a covalent intermediate failed, and a mechanism (see following section) favoring noncovalent enzyme–substrate interme-diates and general acid–general base catalysis is now favored for aspartic proteases

The Mechanism of Action of Aspartic Proteases

A crucial datum supporting the general acid–general base model is the pH depen-dence of protease activity (Figure 14.23) For many years, enzymologists hypothesized that the aspartate carboxyl groups functioned alternately as general acid and general base This model requires that one of the aspartate carboxyls be protonated and one

be deprotonated when substrate binds (This made sense, because X-ray diffraction data on aspartic proteases had shown that the active-site structure in the vicinity of the two aspartates is highly symmetric.) However, Stefano Piana and Paolo Carloni re-ported in 2000 that molecular dynamics simulations of aspartic proteases were con-sistent with a low-barrier hydrogen bond involving the two active-site aspartates This led to a new mechanism for the aspartic proteases (Figure 14.24) that begins with Piana and Carloni’s model of the LBHB structure of the free enzyme (state E) In this model, the LBHB holds the twin aspartate carboxyls in a coplanar conformation, with the catalytic water molecule on the opposite side of a ten-atom cyclic structure Following substrate binding, a counterclockwise flow of electrons moves two pro-tons clockwise and creates a tetrahedral intermediate bound to a diprotonated enzyme form (FT) Then a clockwise movement of electrons moves two protons

(b) (a)

(b)

FIGURE 14.22 Structures of (a) HIV-1 protease, a dimer

(pdb id  7HVP), and (b) pepsin, a monomer Pepsin’s

N-terminal half is shown in red; C-terminal half is

shown in blue (pdb id  5PEP).

0

pH

Pepsin

(a)

pH

HIV protease

(b)

FIGURE 14.23 pH-rate profiles for (a) pepsin and (b) HIV protease.(Adapted from Denburg, J., et al., 1968 The effect of

pH on the rates of hydrolysis of three acylated dipeptides by pepsin Journal of the American Chemical Society 90:479–486; and

Hyland, J., et al., 1991 Human immunodeficiency virus-1 protease 2 Use of pH rate studies and solvent kinetic isotope effects

counterclockwise and generates the zwitterion intermediate bound to a

monopro-tonated enzyme form (ET) Collapse of the zwitterion cleaves the CON bond of

the substrate Dissociation of one product leaves the enzyme in the diprotonated

FQ form Finally, deprotonation and rehydration lead to regeneration of the

ten-atom cyclic structure, E

What is the purpose of the low-barrier hydrogen bond in the aspartic protease

mechanism? It may be to disperse electron density in the ten-atom cyclic structure,

accomplishing rate acceleration by means of “hydrogen tunneling” (Figure 14.25)

The barrier between the ES and ET states of Figure 14.24 is imagined to be large,

and the state FT may not exist as a discrete intermediate but rather may exist

tran-siently to facilitate conversion of ES and ET

The AIDS Virus HIV-1 Protease Is an Aspartic Protease

Recent research on acquired immunodeficiency syndrome (AIDS) and its causative

viral agent, the human immunodeficiency virus (HIV-1), has brought a new

aspar-tic protease to light HIV-1 protease cleaves the polyprotein products of the HIV-1

genome, producing several proteins necessary for viral growth and cellular

infec-tion (Figure 14.26) HIV-1 protease cleaves several different peptide linkages For

example, the protease cleaves between the Tyr and Pro residues of the sequence

Ser-Gln-Asn-Tyr-Pro-Ile-Val, which joins the p17 and p24 HIV-1 proteins

The HIV-1 protease is a remarkable viral imitation of mammalian aspartic

pro-teases: It is a dimer of identical subunits that mimics the two-lobed monomeric

structure of pepsin and other aspartic proteases The HIV-1 protease subunits are

99-residue polypeptides that are homologous with the individual domains of the

monomeric proteases Structures determined by X-ray diffraction studies reveal that

the active site of HIV-1 protease is formed at the interface of the homodimer and

consists of two aspartate residues, designated Asp25and Asp25, one contributed by

S O

C

O

O

R

O–

N R 

R 

C

–

O

C O

H

O

C O

O +

+

– –

O

C O

O

C

O +

+

– –

R  NH 3

H2O

N

C

O

O +

+

–

–

O

C O

O

–

R

R

R 

H N

C O

O

R 

H N

C O

O

R 

H N

COO– R

FIGURE 14.24 Mechanism for the aspartic proteases The letter titles describe the states as follows: E represents

the enzyme form with a low-barrier hydrogen bond between the catalytic aspartates, F represents the enzyme

form with both aspartates protonated and joined by a conventional hydrogen bond, S represents bound

sub-strate, T represents a tetrahedral amide hydrate intermediate, P represents bound carboxyl product, and

Q represents bound amine product This mechanism is based in part on a mechanism proposed by Dexter

Northrop, a distant relative of John Northrop, who had first crystallized pepsin in 1930 (Northrop, D B., 2001 Follow

the protons: A low-barrier hydrogen bond unifies the mechanisms of the aspartic proteases Accounts of Chemical Research

34:790–797.)The mechanism is also based on data of Thomas Meek (Meek, T D., Catalytic mechanisms of the aspartic

proteinases In Sinnott, M., ed, Comprehensive Biological Catalysis: A Mechanistic Reference, San Diego: Academic Press, 1998.)

E+P+Q

E + S ES ET

Reaction coordinate

FIGURE 14.25 Energy level diagram for the aspartic pro-tease reaction, showing ground-state hydrogen tunneling (arrow), with consequent rate acceleration.

each subunit (Figure 14.27) In the homodimer, the active site is covered by two identical “flaps,” one from each subunit, in contrast to the monomeric aspartic pro-teases, which possess only a single active-site flap Enzyme kinetic measurements by Thomas Meek and his collaborators at SmithKline Beecham Pharmaceuticals have shown that the mechanism of HIV-1 protease is very similar to those of other aspar-tic proteases

Chorismate Mutase: A Model for Understanding Catalytic Power and Efficiency

Direct comparison of an enzyme reaction with the analogous uncatalyzed reaction

is usually difficult, if not impossible There are several problems: First, many enzyme-catalyzed reactions do not proceed at measurable rates in the absence of the enzyme Second, many enzyme-catalyzed reactions involve formation of a cova-lent intermediate between the enzyme and the substrate Third, a reaction occur-ring in an enzyme active site might proceed through a different transition state than

the corresponding solution reaction Chorismate mutase is a happy exception to all

these potential problems First, although the rate of this reaction is more than a mil-lion times faster on the enzyme, the uncatalyzed solution reaction still proceeds at reasonable and measurable rates Second, the enzyme reaction does not employ a covalent intermediate What about the transition states for the catalyzed and un-catalyzed reactions? Chorismate mutase acts in the biosynthesis of phenylalanine and tyrosine in microorganisms and plants It involves a single substrate and cat-alyzes a concerted intramolecular rearrangement of chorismate to prephenate In

mRNA

Translation

Protease

gag–pol polyprotein

p24 p66/51 (reverse transcriptase)

p7 p6

FIGURE 14.26 HIV mRNA provides the genetic

informa-tion for synthesis of a polyprotein Proteolytic cleavage

of this polyprotein by HIV protease produces the

indi-vidual proteins required for viral growth and cellular

infection.

ACTIVE FIGURE 14.27 (left) HIV-1

pro-tease complexed with the inhibitor Crixivan (red) made

by Merck The flaps (residues 46–55 from each subunit)

covering the active site are shown in green, and the

active-site aspartate residues involved in catalysis are

shown in light purple (right) The close-up of the active

site shows the interaction of Crixivan with the carboxyl

groups (yellow) of the essential aspartate residues (pdb

id 1HSG) Test yourself on the concepts in this

figure at www.cengage.com/login.